One-Year Follow-Up of COVID-19 Patients Indicates Substantial Assay-Dependent Differences in the Kinetics of SARS-CoV-2 Antibodies

ABSTRACT Determination of antibody levels against the nucleocapsid (N) and spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are used to estimate the humoral immune response after SARS-CoV-2 infection or vaccination. Differences in the design and specification of antibody assays challenge the interpretation of test results, and comparative studies are often limited to single time points per patient. We determined the longitudinal kinetics of antibody levels of 145 unvaccinated coronavirus disease 2019 (COVID-19) patients at four visits over 1 year upon convalescence using 8 commercial SARS-CoV-2 antibody assays (from Abbott, DiaSorin, Roche, Siemens, and Technoclone), as well as a virus neutralization test (VNT). A linear regression model was used to investigate whether antibody results obtained in the first 6 months after disease onset could predict the VNT results at 12 months. Spike protein-specific antibody tests showed good correlation to the VNT at individual time points (rS, 0.74 to 0.92). While longitudinal assay comparison with the Roche Elecsys anti-SARS-CoV-2 S test showed almost constant antibody concentrations over 12 months, the VNT and all other tests indicated a decline in serum antibody levels (median decrease to 14% to 36% of baseline). The antibody level at 3 months was the best predictor of the VNT results at 12 months after disease onset. The current standardization to a WHO calibrator for normalization to binding antibody units (BAU) is not sufficient for the harmonization of SARS-CoV-2 antibody tests. Assay-specific differences in absolute values and trends over time need to be considered when interpreting the course of antibody levels in patients. IMPORTANCE Determination of antibodies against SARS-CoV-2 will play an important role in detecting a sufficient immune response. Although all the manufacturers expressed antibody levels in binding antibody units per milliliter, thus suggesting comparable results, we found discrepant behavior between the eight investigated assays when we followed the antibody levels in a cohort of 145 convalescent patients over 1 year. While one assay yielded constant antibody levels, the others showed decreasing antibody levels to a varying extent. Therefore, the comparability of the assays must be improved regarding the long-term kinetics of antibody levels. This is a prerequisite for establishing reliable antibody level cutoffs for sufficient individual protection against SARS-CoV-2.

The authors study an interesting area, although data are now available from several different studies of a similar nature. The reviewer has raised important issues around whether reinfection occurred, the differences in antibody persistence between patients with single and multiple morbidities, and data on the type of infecting virus (ie what VOC was likely to be infecting these patients). I agree these are important issues that should be addressed with a resubmitted manuscript, that would then be considered as modifications of the existing manuscript. The authors are encouraged to address these.
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The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. This longitudinal study investigated level of humoral immune response in COVID-19 patients for up to one year following SARS-CoV-2 infection, thereby evaluating 8 commercially available anti-spike and anti-nucleocapsid antibody assays, as well as virus neutralisation test. I only have a few minor comments.
(1) The length of study is relatively long, and re-infection is possible in study participants during one year follow-up. How was reinfection being monitored during the study period?
(2) Live virus neutralisation test was validated using the WHO reference panel and reference standard for anti-SARS-CoV-2 antibodies. Good correlation was observed between neutralising antibody titres determined by virus neutralisation test and WHO assigned International Units. It would be interesting to determine the performance of 8 commercial SARS-CoV-2 antibody assays (in terms of correlation with WHO assigned International Units), using reference panel and reference standards.
(3) Clinical data including gender, age, severity of acute COVID-19 , polymorbidity, etc. were obtained for each participant. Analysis of antibody levels and COVID-19 disease severity in polymorbidity subgroup could not be found.

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Reply to Reviewers
Reviewer: 1 This longitudinal study investigated level of humoral immune response in COVID-19 patients for up to one year following SARS-CoV-2 infection, thereby evaluating 8 commercially available anti-spike and anti-nucleocapsid antibody assays, as well as virus neutralisation test. I only have a few minor comments.

The length of study is relatively long, and re-infection is possible in study participants during one year follow-up. How was re-infection being monitored during the study period?
Reply: We agree that re-infection could have been an issued during the observation period of one year. Participants have been asked for symptoms associated with a re-infection at each study visit. No patient had to be excluded due to a symptomatic re-infection. Asymptomatic re-infections can formally not be excluded as regular PCR test have not been performed for monitoring within the study. However, individual antibody levels did not indicate an immune reaction to a re-infection. A paragraph on this minor limitation has been added to the discussion section of the revised manuscript.

Live virus neutralisation test was validated using the WHO reference panel and reference standard for anti-SARS-CoV-2 antibodies. Good correlation was observed between neutralising antibody titres determined by virus neutralisation test and WHO assigned International Units. It would be interesting to determine the performance of 8 commercial SARS-CoV-2 antibody assays (in terms of correlation with WHO assigned International Units), using reference panel and reference standards.
Reply: We thank the reviewer for raising this point. We measured dilutions of the WHO reference standard with all 6 quantitative assays used in our study. Data are shown in Table S6 of the revised manuscript. In brief, the WHO standard showed a good agreement between the assays. A comment that the observed differences in the long-term follow-up samples could not be explained due to differences in the calibration according to the WHO standard has been added in the revised manuscript.
3. Clinical data including gender, age, severity of acute COVID-19 , polymorbidity, etc. were obtained for each participant. Analysis of antibody levels and COVID-19 disease severity in polymorbidity subgroup could not be found.
Reply: We agree that data on multimorbidity had not been analysed in particular. A stratification of antibody levels according to multimorbidity has been added as additional subpanel in Figure S1. The overlap between multimorbidity and COVID-19 disease severity according to WHO has been added as Table S8 in the revised manuscript. Re: Spectrum00597-22R1 (One year follow-up of COVID-19 patients indicates substantial assay-dependent differences in the kinetics of SARS-CoV-2 antibodies) Dear Dr Hoermann: The paper is now suitable for publication with responses to the reviewers from the authors.
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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data. If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record. If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.